This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Human Pol ? is composed of four subunits and its activity is enhanced dramatically by interaction with PCNA. The largest p125 subunit has both polymerase and 3? to 5? exonuclease activities and mutations at the p125 active sites increase genomic instability and accelerate tumorigenesis.[8,9] The remaining three subunits, p50, p68, and p12 are thought to play a regulatory role, stimulate the polymerase activity of p125 by mediating additional interactions with PCNA, and stabilize the entire Pol ? complex. p50 serves as a scaffold for the assembly of Pol ? by interacting simultaneously with all of the other three subunits. Interestingly, many essential functions of Pol ?, including the regulation of replication, trans-lesion DNA synthesis and break-induced recombination, are mediated by the third subunit. Recently we reported the crystal structure of the complex between the second p50 subunit and the 144 amino acids N-terminal domain of the third p66 subunit (p66N) of human Pol ?. However, interactions of p50/p66 with p125 and p12 subunits, as well as DNA, remain unknown. We also succeeded in prepared of soluble monodisperse samples of p125, p50/p66, and p12, and confirmed the in vitro reconstitution of four-subunit Pol ?. Our long-term goal is to reveal the structure-based molecular mechanisms of human Pol ? function. Our short-term goal is to reveal the three-dimensional organization of Pol ? complex and locate the exact position of each subunit and map the areas involved in intersubunit interactions. To reach this goal we will combine cryo EM experiments with X-ray crystallography of individual domains and confirm our conclusions with biochemical and functional experiments. The data will allow as the positioning of the recently solved X-ray structure of human p50/p66 into the electron density maps obtained by Cryo EM, thus giving more complete information about the intersubunit interactions within Pol ?. We will test the defined subunit interactions with mutations and yeast two-hybrid assay.